Coupled Abscription PCR Signaling (CAPS)

The sensitivity of RiboMed’s proprietary detection technology CAPS (Coupled Abscription PCR Signaling), together with its ability to detect small DNA targets (less than 50 bp), make it ideal for the detection of biomarkers in bodily fluids (liquid biopsies) and in Formalin Fixed Paraffin Embedded (FFPE) tissues. DNA methylation profiles have been successfully obtained from FFPE brain tumors stored for over 25 years. Gene specific DNA methylation measurements have been successful with six genomic copies of DNA, or three cell equivalents.

Abscription (Abortive Transcription) is an extremely robust isothermal signal generation reaction that involves the reiterative synthesis of short RNA Abscripts® (abortive transcripts), which are produced by a proprietary RNA polymerase (Abscriptase®), to generate hundreds to thousands of detectable and quantifiable abscripts per target molecule per minute (reiterative synthesis, see link below). Abscripts are generated from an artificially created start site for Abscription, called an Abortive Promoter Cassette (APC). Each APC encodes a different unique abscript of defined RNA sequence (3 to 12 nt long). Abscripts can be detected by a variety of methods, depending on their size, sequence, mass, polarity or other physical properties.

Abscription can be used to detect most types of molecular biomarkers (DNA, RNA, protein, DNA methylation, mutations, deletions). APCs are linked to targeted molecules for detection, therefore multiplexing can be achieved by attaching a different APC to each biomarker target in a sample. Abscription can be used to detect nucleic acids and their modifications with unparalleled sensitivity by coupling it to a target amplification system, such as PCR or isothermal amplifications systems such as LAMP. Abscription can be used alone for detection of protein by attaching an APC to an antibody, as shown in this video link (shows detection of a toxin protein from a pathogen). The Abscription step is independent of the target type or sequence and works the same for nucleic acid detection. The sequence of the Abscript is NOT related to the sequence of the nucleic acid target, but is encoded by the APC. Target specificity is determined by antibodies for proteins and by target specific primers for nucleic acids.

For the detection of nucleic acids (DNA, RNA, DNA methylation, mutations), Abscription is partnered with target specific amplification in a process called CAPS (Coupled Abscription PCR Signaling), which is 2 to 3 orders of magnitude more sensitive than Taqman qPCR-based detection. APCs are attached to the target nucleic acid during an initial PCR reaction with target-specific primers containing an inactive, single-stranded APC overhang. Upon amplification, the ss APC is converted to an active ds APC containing the Abscriptase binding site. Addition of Abscriptase and the dinucleotide initiator and NTPs encoded by the attached APC, a specific abscript is reiteratively synthesized. In the fluorescent based assay, each abscript hybridizes to the 3′ end of the loop of a quenched molecular beacon. The CAPS polymerase extends the abscript to the end of the beacon, resulting in the separation of the beacon’s fluorescent tag and quencher. Fluorescence is produced only in the presence of target.

For multiplexing, different APCs can be attached to different targets, with each abscript priming the opening of a different molecular beacon, each containing a different fluorophore.